The use of peptides and peptide-like molecules as tags for attaching to proteins and other entities is an important tool in molecular biology. Such peptide tags can allow the detection, purification and analysis of a particular protein or entity or can be used for the specific targeting of the tagged protein or entity. Thus, peptide tags, which may be attached to a protein of interest using recombinant DNA methods (e.g. by operably linking the nucleotide sequence encoding the peptide tag with the gene encoding the protein of interest and expressing the protein product), usually have the ability to bind to a binding partner. This binding may allow the detection of the protein of interest if the binding partner is detectable, e.g. is an antibody or is conjugated to a detectable entity, or can allow purification of the protein of interest if the binding partner is, for example, immobilised to a solid support. Thus, the use of peptide tags which are capable of associating with a binding partner can provide a means for manipulating or analysing a target protein or entity; this analysis can be used to determine the size, abundance, location in the cell or organism, and the interactions of the tagged protein.
There are a number of different peptide tag systems which are known in the art and which are available commercially, for example, c-myc, FLAG, HA, His6, T7-Tag, Strep-Tag, Avi-Tag to name a few. As indicated above, such peptide tag systems are widely used for detecting, characterising and purifying proteins, where the use of a peptide tag may for example abrogate the need to develop an antibody to the protein of interest, which is a time-consuming and expensive process that is not always successful. The small size of the peptide tag (usually 5-15 amino acids in length) generally has no effect on the biological function of the protein of interest to which it is attached. However, one major problem with the use of peptide tags of the art is the instability of their interactions with their binding partners, with affinity often micromolar and rarely better than nanomolar, relating to the limited accessible surface area and the intrinsic flexibility of the peptides. The peptide flexibility places a large entropic cost on forming a well defined binding interaction. There is therefore a lack of peptide tag/binding partner systems in the art which provide high affinity or irreversible binding and which would be useful to improve the sensitivity of protein detection, the efficiency and yield of protein purification and to provide a rigid link to hold together proteins which are subject to high forces.